A large number of organic chemicals are currently derived from petrochemical feedstocks. There is a growing interest in producing many of these petrochemical-derived organic compounds through biological fermentation processes using renewable feedstock. The list of organic compounds that can be derived from renewable feedstock includes 1,4-diacids (succinic, fumaric, malic, glucaric, malonic, and maleic), 2,5-furan dicarboxylic acid, propionic acid, 3-hydroxy propionic acid, aspartic acid, glucaric acid, glutamic acid, itaconic acid, levulinic acid, 3-hydroxybutyrolactone, glycerol, sorbitol, xylitol, arabinitol. and butanediols such as 1,4 butanediol, 1,3-butanediol, and 2,3-butanediol. Besides these compounds, many other types of organic compounds including, but not limited to, amino acids, vitamins, alcohols (such as ethanol, n-propanol, isopropanol, n-butanol, isobutanol, and higher alcohols), fatty acids, esters of fatty acids, hydrocarbons, isoprenoids, turpenes, carotenoids, and amines can also be produced using renewable feedstocks. Any such compound shall be referred to herein as a “desired compound”. However, for most of these desired compounds, a commercially attractive fermentation process that uses sucrose as a carbon source has not yet been developed.
Among these organic compounds that can be derived from renewable feedstock, succinic acid deserves a special mention. A number of microbial species including Escherichia coli, Corynebacterium glutamicum and Brevibacteium flavum, Mannhemia succiniproducens, Basfia succiniproducens and Anaerobiospirilum succiniproducens have been genetically manipulated for use as a biocatalyst in the production of succinic acid using renewable feedstocks. These biocatalysts have been shown to produce succinic acid in very high yield and productivity. Currently, succinic acid production using these biocatalysts is carried out using dextrose as the source of organic carbon.
There is a need to develop processes that use cheaper renewable feedstock in order to make the biological production of succinic acid to be competitive with succinic acid production from petrochemical feedstocks using purely chemical processes. Efforts are under way to use carbon sources derived from the hydrolysis of cellulosic materials as the feedstock for succinic acid production using a biocatalyst. However, the techniques for producing feedstock from cellulosic materials for biological fermentation are far from perfected at this time. Thus there is an immediate need for a cost-effective feedstock suitable for biological production of succinic acid. The price of various sugars varies with time and geographic location. In many part of the world, sucrose is less expensive than glucose most or all of the time, especially in tropical areas where sugarcane thrives.
Molasses derived from the sugarcane processing represents a cost-effective feedstock for any industrial fermentation. Molasses is inexpensive, readily available, and abundant. Molasses is rich in sucrose and there is a need to develop biocatalysts with the ability to use sucrose as the source of carbon in the fermentative production of succinic acid. In some cases, it will be preferable to use purified sucrose in the fermentation as a carbon source, for example when the fermentation product of interest must be highly purified, and where it is more cost effective to remove the non-sugar compounds from the molasses prior to fermentations. Purified sucrose in the form of “table sugar” has been produced for many years by well known methods, and material equivalent to this can also be used as carbon source in the present invention. In addition, molasses or sucrose utilization by biocatalysts has wider industrial significance as molasses or sucrose can be used to produce a wide variety of organic chemicals as well as whole cell mass. Thus the present invention has broader application in the fermentation industry in general beyond the fermentative production of succinic acid. A sucrose-containing medium can comprise juice or molasses derived from sugar cane, sugar beet, sweet sorghum, or any other sucrose-containing plant material.
A mechanism for the transport of sucrose and other sugars that is frequently found in bacteria is based on the phosphotransferase system (PTS). PTS is composed of two-energy-coupling proteins, Enzyme I and HPr, and several sugar-specific Enzyme II proteins or protein complexes, which typically consist of three protein domains, EIIA, EIIB and EIIC. The organization of the EII domains differs between bacteria. EII may consist of a single fused protein or different fused and unfused domains. The translocation of the specific sugar through the membrane is facilitated by the integral membrane domain. However, it is the complex of the three enzyme domains or proteins, functioning together, which brings about the transport and phosphorylation of the sugar substrate, resulting in an intracellular pool of phosphorylated carbohydrate (Neidhardt and Curtiss, 1996).
There are alternative mechanisms for bacterial sucrose transport in addition to the PTS. These PTS-independent sugar permeases facilitate sucrose accumulation without chemical modifications. They include solute-cation symport systems, such as the ScrT symporter in the sucrose operon in Bifidobacerium lactis and the CscB transporter of E. coli W strain. These sucrose-specific transport systems are generally clustered with the catabolic and regulatory genes in various arrangements in different bacteria.
There are several examples in the literature that describe the cloning of sucrose utilization genes and the installation of such genes into organisms that did not natively contain said genes. There are examples of cloning and transfer of both PTS-dependent and PTS-independent sucrose utilization genes. However, each of these examples has at least one feature that makes them undesirable for use in a commercial setting. For example, maintenance of sucrose utilization genes on replicating plasmids (such as the pScr1 plasmid described in US Patent Application Publication No. 2008/0274526 A1) is not desirable because plasmids can be unstable (Shukla et al., 2004) or lost during the many generations of large scale growth, and they often require presence of an antibiotic for maintenance. Also, expression of a gene from a multicopy plasmid can be excessive, leading to waste of energy and materials, or inhibition of growth.
The chromosomal PTS-independent sucrose utilization genes cscAKB from E. coli EC3132 have been integrated into the chromosome of E. coli K-12 derived strains (U.S. Pat. No. 6,960,455). But this particular operon is known to be relatively inefficient at conferring sucrose utilization, especially at low sucrose concentrations, where the doubling time was 20 hours (1200 minutes) (Bockmann et al., 1992). A homology comparison of the DNA sequence of the cscRAKB operon region from EC3132 with that of an efficient sucrose utilizer, E. coli ATCC 9637 (Shukla et al., 2004) using the Megalign program of DNASTAR software revealed a similarity index of 98.1. Thus there were many differences between the two csc operons, and these differences are scattered throughout the operon. Many of the point mutations in the EC3132 operon cause non-conservative changes in protein sequences, and some of the mutations were in promoter regions, so it must be the case that the operon from EC3132 is simply defective due to one or more of these mutations. Faster growing spontaneous mutants with a doubling time on sucrose of down to 50 minutes could be isolated from EC3132, but when the mutated cscAKB operons were installed in a K-12 strain, the doubling time on sucrose medium increased back up to 75 minutes at best (Jahreis et al., 2002). None of the mutated csc operons from EC3132 changed the DNA sequence to be more like the efficient operon of ATCC 9637. As such, it appears that the operon disclosed by Jahreis et al (2002) is not as desirable as that of ATCC 9637.
Another approach that has been proposed to enable sucrose utilization is to simply engineer a strain to secrete invertase (Lee et al., 2010a). In this case the invertase from Mannheimia succiniproducens was shown to be superior to the invertase from E. coli W, teaching away from the use of the E. coli W invertase. Moreover, in bacteria, this approach is inherently less efficient than importing the sucrose before cleaving it, since after cleavage to glucose and fructose by invertase, two sugar molecules must be imported rather than just one, and this requires more energy to be expended. Moreover, in this prior art example, during fermentation starting with 20 g/l sucrose, external fructose accumulated to over 10 g/l, and it took 50 hours for this external fructose to be completely consumed (Lee et al., 2010b). Thus, although a sucrose utilizing strain was disclosed, the system had performance characteristics that were not commercially attractive. Secretion of fructose by E. coli strains grown on sucrose is a general problem, and the fructose that remains at the end of a desirable fermentation time, which is often 48 hours or less, is undesirable. Thus, there is a need for microorganisms that can ferment sucrose to a desirable product at a commercially attractive titer, which is usually more than 20 grams per liter, without leaving more than 2 grams per liter of fructose remaining after a fermentation time of 48 hours or less.
E. coli strains SZ63 and SZ85 were previously engineered to produce optically pure D-lactate from hexose and pentose sugars. To expand the substrate range to include sucrose, a cscR′AKB operon was cloned and characterized from E. coli KO11, a derivative of E. coli W (Shukla et al., 2004). The resulting plasmid-borne operon was functionally expressed in SZ63 but was unstable in SZ85.
U.S. Pat. No. 6,960,455 discloses a method of producing amino acids using E. coli K-12 derived strains transduced with a cscRAKB operon from strain EC3132 that is located at the dsdA locus in the chromosome. As a consequence of insertion of cscRKAB operon at this location in the chromosome, the resulting strains cannot catabolize D-serine. As pointed out above, the csc genes used in this prior art were derived from a defective EC3132 operon, which grows poorly on low concentrations of sucrose (Bockmann et al., 1992; Jahreis et al, 2002). The resulting strains contain the cscR gene, which encodes a repressor, so the strains are expected to be suboptimal for sucrose utilization, especially in the presence of glucose, which would be expected to cause repression of the cscAKB genes (Jahreis et al., 2002; Shukla et al., 2004). Molasses usually contains some glucose. There was no attempt mentioned to correct any of the defects inherent in the csc operon of EC3132 in U.S. Pat. No. 6,960,455. Moreover, production of chemicals other than amino acids, such as succinate were not mentioned in this patent.
U.S. Pat. No. 7,179,623 discloses a strain constructed from a sucrose non-assimilative strain wherein the said strain harbors sucrose PTS genes from E. coli VKPM B-7915 (scrKYABR genes), but again, this patent does not mention production of chemicals other than amino acids, and the PTS-dependent system claimed in this disclosure is not optimal for production of chemicals that are derived from phosphoenol pyruvate (PEP).